Neuroscience Letters, 147 (1992) 121-124
121
© 1992ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$05.00 NSL 09108
Ultrastructural study of skein-like inclusions in anterior horn neurons of patients with motor neuron disease Shoichi Sasaki a n d Shoichi M a r u y a m a Department of Neurology, Neurological Institute, Tokyo Women's Medical College, Tokyo (Japan)
(Received21 July 1992;Revised version received 1 September 1992;Accepted2 September1992) Key words: Motor neuron disease; Skein-likeinclusion; Lewy body-likehyaline inclusion; Bunina body; Ultrastructure
We investigatedthe ultrastructure of skein-likeinclusions(SI) in 11 patients with motor neuron disease. SI mainlyconsistedof bundles of filaments. Each filament was approximately 15-25 nm in diameter and had a tubular profile on transverse sections. SI occasionallyappeared to contain electron-densematerial similar to Bunina bodies (BB),and in clusters of SI, electron-densematerial closelyresemblingBB with vesicleswerescattered among the bundles of filaments or were directlyattached to the filaments, suggesting a close relationship between SI and BB. We also infer that SI might be the precursor of Lewy body-likehyaline inclusions (LBHI), on the basis of the frequent presenceof in-betweenstructures, the coexistence of SI and LBHI, the detection of tubular profiles(componentof SI) in LBHI, the far greater frequencywith which SI were observedcompared with LBHI, and the occasional connectionbetween ubiquitin-positiveSI and LBHI observedin preceding immunocytochemicalstudies.
Recently, ubiquitin-immunoreactive intraneuronal inclusions, such as hyaline inclusions [1, 2], Lewy body-like hyaline inclusions (LBHI) [3-5, 8-11, 13, 14] and skeinlike inclusions (SI) [3, 4, 6-9, 11, 14, 15] have come to be considered pathological hallmarks of m o t o r neuron disease (MND). They appear to be quite specific to M N D [4, 6, 8, 9, 11, 14, 15] as is the case for Bunina bodies (BB). Structures seemingly identical to the skein-like filamentous arrays first described by Leigh et al. [4] have been subsequently described by various terms such as loose filamentous inclusions [6, 8], focally aggregated filamentous structures [11], thread-like structures [3, 13], and skein-like inclusions [7, 9, 15]. They have been reported in classical ALS [6, 11], lower m o t o r neuron disease [3, 9, 14], Guamanian ALS [8], and familial ALS [10]. However, little information is available about the ultrastructure of SI [9, 11, 14], and the relationship between these structures and LBHI or BB still remains controversial. In this study, we investigated SI to elucidate their ultrastructural details and their possible relationship with LBHI and BB. We studied 11 patients with SI (aged 49-83 years; mean age 61.7 years) from among 27 M N D patients (Table I). For electron microscopic examination, the Correspondence: S. Sasaki, Department of Neurology, Neurological Institute, Tokyo Women's Medical College, Tokyo, 162, Japan. Fax: (81) 3 5269 7324.
lumbar (L1_5) levels o f the spinal cords were fixed in 2% glutaraldehyde with phosphate buffer (pH 7.40). After fixation, the anterior horns at each level were then sectioned transversely, postfixed in 1% osmium tetroxide for several hours, dehydrated, and embedded in epoxy resin. Each block was cut into serial semi-thin sections (about 1 p m thick). These sections were stained with Toluidine blue. Appropriate portions of the sections were cut into ultrathin sections. These were then stained with uranyl acetate and lead citrate. The same procedure was applied to 11 control patients (aged 44-81 years; mean age 59.5 years) who died without having any known neurological disease. Autopsy was performed on all M N D and control patients within 3 h of death. Electron microscopy showed that SI consisted basically of a bundle of compactly packed filaments running parallel to the longitudinal axis without fine granules (Fig. 1). Each filament measured approximately 15-25 nm in diameter and was tubular on transverse sections. A bundle of these grouped tubular filaments was often surrounded by a unit membrane (Fig. 1). SI occasionally appeared to contain electron-dense material (Fig. 2). Sometimes SI showed a combination of fine filaments (neurofilaments) and thicker linear structures (tubules) (Fig. 3). LBHI were composed of randomly arranged filaments associated with fine granules. In addition to SI and LBHI, we often observed in-between or LBHI-like structures (Figs. 4-6). They consisted of dense linear
122 TABLE I NEUROPATHOLOGICAL FINDINGS SI, skein-like inclusion; LBHI, Lewy body-like hyaline inclusion; BB, Bunina body; -, absent; +, mild; ++, moderate; +++, high. Frequency of appearance
Case Age/sex Clinical Degreeof No. course anterior (months) horn cell loss
1 2 3 4 5 6 7 8 9 10 lI
75/M 83/M 49/M 62/M 67/F 67/M 49/M 62/M 63/F 64/M 78/F
6 10 16 22 24 28 29 30 42 65 68
+ + ++ + + ++ + ++ + + ++
SI
LBHI
BB
++ +++ + ++ + +++ + ++ ++ ++
+++ +++ + + + +++ + + +
+ +++ + ++ ++ ++ + ++ +++ ++
structures, frequently c o n t a i n i n g finer filaments (intermediate filaments) (Figs. 4 a n d 5) a n d occasionally elect r o n - d e n s e material; some of them closely resembled BB that consist of electron-dense g r a n u l a r or a m o r p h o u s material frequently with vesicles a n d t u b u l a r structure (Fig. 5), a n d m o r e rarely h a d large electron-dense central cores (Fig. 6). W h e r e the r a n d o m l y - a r r a n g e d thicker filam e n t s were clustered, there were also scattered clumps of electron-dense g r a n u l a r or a m o r p h o u s material closely resembling BB with vesicles, a n d these were sometimes m i n g l e d with the thicker filaments (Fig. 7). SI were freq u e n t l y observed at the periphery o f or inside L B H I , a n d even s u r r o u n d e d some of the L B H I (Fig. 8). T u b u l a r
Fig. 1. A skein-like inclusion consists of bundles of compactly packed filaments running parallel to the longitudinal axis without fine granules. Each filament is approximately 15-25 nm in diameter, x 51,300.
Fig. 2. A skein-like inclusion appears to contain electron-dense material. × 51,300.
profiles were sometimes seen in the L B H I o n cross-sections. SI were m u c h more c o m m o n t h a n L B H I in the anterior horn neurons. The control subjects showed n o b u n d l e s of filaments c o r r e s p o n d i n g to SI in their a n t e r i o r h o r n n e u r o n s . It remains controversial whether SI have any relationship to BB. Recently, M u r a y a m a et al. [11] reported the existence of BB-related structures consisting of b u n d l e s of coated filaments (12 n m in diameter) as possible precursors of BB, a n d these a p p e a r to be identical to the b u n d l e s of t u b u l a r filaments detected in the present study. O n the other h a n d , there are some reports that oppose this conclusion. It has been p o i n t e d out that SI differ from BB b o t h electron microscopically a n d imm u n o c y t o c h e m i c a l l y [9, 1 1, 14]. U n l i k e SI, BB consist of electron-dense material with vesicles a n d t u b u l a r structures, a n d occasionally c o n t a i n cytoplasmic organelles such as n e u r o f i l a m e n t s a n d m i t o c h o n d r i a [12]. I n addition, SI are u b i q u i t i n - p o s i t i v e a n d BB are u b i q u i t i n - n e g -
Fig. 3. A skein-likeinclusion shows a combination of fine filaments and thicker linear structures, x 51,300.
123
Fig. 4. An in-between structure of SI and LBHI which is composed of bundles of thicker filaments containing finer filaments (neurofilaments), x 51,300.
Fig. 5. An in-between structure of SI and LBHI (or LBHI-like structure) which consists of bundles of thicker filaments containing finer filaments (intermediate filaments) and electron-dense material resembling Bunina bodies, x 19,000.
Fig. 7. Electron-dense material closely resembling Bunina bodies with vesicles is scattered among clusters of skein-like inclusions, x 15,200.
ative [3, 14]. In the present study, m a n y SI were indeed f o u n d at separate locations f r o m the BB. However, SI occasionally contained electron-dense material similar to BB, and electron-dense material closely resembling BB were not u n c o m m o n l y mingled with SI. These findings suggest a close relationship between SI and the f o r m a tion o f BB. The relationship between SI and L B H I has also been debated. Some investigators report that the frequency o f SI is not correlated with the presence or absence o f L B H I [9, 11]. However, the present study frequently showed in-between structures and the coexistence o f SI and L B H I . Tubular profiles ( c o m p o n e n t o f SI) were sometimes observed in L B H I , and SI were far m o r e frequently seen c o m p a r e d with L B H I . These findings, taken together with the fact that ubiquitin-positive SI are occasionally connected with ubiquitin-positive L B H I in imm u n o c y t o c h e m i c a l studies [3, 13, 14], seem to indicate a close relationship between these two structures including a possibility that SI m a y be the precursor o f L B H I .
Fig. 6. A LBHI-like structure containing a large central electron-dense core. × 19,000. Fig. 4-6. In-between structures of skein-like inclusions (SI) and Lewy body-like hyaline inclusions (LBHI), or LBHI-like structures.
Fig. 8. Coexistence of LBHI and skein-like inclusions, x 9,500.
124 1 Hirano, A., Kurland, L.T. and Sayre, G.P., Familial amyotrophic lateral sclerosis. A subgroup characterized by posterior and spinocerebellar tract involvement and hyaline inclusions in the anterior horn cells, Arch. Neurol., 16 (1967) 232-243. 2 Hirano, A., Nakano, I., Kurland, L.T., Mulder, D.W., Holley, P.W. and Accomanno, G., Fine structural study of neurofibrillary changes in a family with amyotrophic lateral sclerosis, J. Neuropathol. Exp. Neurol., 43 (1984) 471-480. 3 Kato, T., Katagiri, T., Hirano, A., Kawanami, T. and Sasaki, H., Lewy body-like hyaline inclusions in sporadic motor neuron disease are ubiquitinated, Acta Neuropathol., 77 (1989) 391-396. 4 Leigh, P.N., Anderton, B.H., Dodson, A., Gallo, J.-M., Swash, M. and Power, D.M., Ubiquitin deposits in anterior horn cells in motor neuron disease, Neurosci Lett., 93 (1988) 197-203. 5 Leigh, P.N., Whitwell, H., Garofalo, O., Buller, J., Swash, M., Martin, J.E., Gallo, J.-M., Weller, R.O. and Anderton, B.H., Ubiquitinimmunoreactive intraneuronal inclusions in amyotrophic lateral sclerosis, Brain, 114 (1991) 775-788. 6 Lowe, J., Lennox, G., Jefferson, D., Morrell, K., McQuire, D., Gray, T., Landon, M., Doherty, F.J. and Mayer, R.J., A filamentous inclusion body within anterior horn neurones in motor neurone disease defined by immunocytochemical localization of ubiquitin, Neurosci. Lett., 94 (1988) 203-210. 7 Martin, J.E., Swash, M. and Schwartz, M.S., New insights in motor neuron disease, Neuropathol. Appl. Neurobiol., 16 (1990) 97-110. 8 Matsumoto, S., Hirano, A. and Goto, S., Ubiquitin-immunoreactive filamentous inclusions in anterior horn cells of Guamanian amyotrophic lateral sclerosis, Acta Neuropathol., 80 (1990) 233 238,
9 Mizusawa, H., Nakamura, H., Wakayama, I., Yen, S.-H. and Hirano, A., Skein-like inclusions in the anterior horn cells in motor neuron disease, J. Neurol. Sci., 105 (1991) 14-21. 10 Murayama, S., Ookawa, Y., Mori, H., Nakano, I., Ihara, Y., Kuzuhara, S. and Tomonaga, M., Immunocytochemicaland ultrastuctural study of Lewy body-like hyaline inclusions in familial amyotrophic lateral sclerosis, Acta Neuropathol., 78 (1989) 143152. 11 Murayama, S., Mori, H., Ihara, Y, Bouldin, T.W., Suzuki, K. and Tomonaga, M., Immunocytochemical and ultrastructural studies of lower motor neurons in amyotrophic lateral sclerosis, Ann. Neurol., 27 (1990) 137-148. 12 Sasaki, S., Hirano, A., Donnenfeld, H. and Nakano, I., An electron microscopic study of Bunina bodies, Neurol. Med., (Tokyo) 19 (1983) 317-324. 13 Sasaki, S., Yamane, K., Sakuma, H. and Maruyama, S., Sporadic motor neuron disease with Lewy body-like hyaline inclusions, Acta Neuropathol., 78 (1989) 555-560. 14 Sasaki, S. and Maruyama, S., Immunocytochemical and ultrastrucrural studies of hyaline inclusions in sporadic motor neuron disease, Acta Neuropathol., 82 (1991) 295-301. 15 Schiffer, D., Autilio-Gambetti, L., Chio, A., Gambetti, P., Giordana, M.T., Gullotta, F., Migheli, A. and Vigliani, M.C., Ubiquitin in motor neuron disease: study at the light and electron microscope, J. Neuropathol. Exp. Neurol., 50 (1991) 463-473.
121
© 1992ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$05.00 NSL 09108
Ultrastructural study of skein-like inclusions in anterior horn neurons of patients with motor neuron disease Shoichi Sasaki a n d Shoichi M a r u y a m a Department of Neurology, Neurological Institute, Tokyo Women's Medical College, Tokyo (Japan)
(Received21 July 1992;Revised version received 1 September 1992;Accepted2 September1992) Key words: Motor neuron disease; Skein-likeinclusion; Lewy body-likehyaline inclusion; Bunina body; Ultrastructure
We investigatedthe ultrastructure of skein-likeinclusions(SI) in 11 patients with motor neuron disease. SI mainlyconsistedof bundles of filaments. Each filament was approximately 15-25 nm in diameter and had a tubular profile on transverse sections. SI occasionallyappeared to contain electron-densematerial similar to Bunina bodies (BB),and in clusters of SI, electron-densematerial closelyresemblingBB with vesicleswerescattered among the bundles of filaments or were directlyattached to the filaments, suggesting a close relationship between SI and BB. We also infer that SI might be the precursor of Lewy body-likehyaline inclusions (LBHI), on the basis of the frequent presenceof in-betweenstructures, the coexistence of SI and LBHI, the detection of tubular profiles(componentof SI) in LBHI, the far greater frequencywith which SI were observedcompared with LBHI, and the occasional connectionbetween ubiquitin-positiveSI and LBHI observedin preceding immunocytochemicalstudies.
Recently, ubiquitin-immunoreactive intraneuronal inclusions, such as hyaline inclusions [1, 2], Lewy body-like hyaline inclusions (LBHI) [3-5, 8-11, 13, 14] and skeinlike inclusions (SI) [3, 4, 6-9, 11, 14, 15] have come to be considered pathological hallmarks of m o t o r neuron disease (MND). They appear to be quite specific to M N D [4, 6, 8, 9, 11, 14, 15] as is the case for Bunina bodies (BB). Structures seemingly identical to the skein-like filamentous arrays first described by Leigh et al. [4] have been subsequently described by various terms such as loose filamentous inclusions [6, 8], focally aggregated filamentous structures [11], thread-like structures [3, 13], and skein-like inclusions [7, 9, 15]. They have been reported in classical ALS [6, 11], lower m o t o r neuron disease [3, 9, 14], Guamanian ALS [8], and familial ALS [10]. However, little information is available about the ultrastructure of SI [9, 11, 14], and the relationship between these structures and LBHI or BB still remains controversial. In this study, we investigated SI to elucidate their ultrastructural details and their possible relationship with LBHI and BB. We studied 11 patients with SI (aged 49-83 years; mean age 61.7 years) from among 27 M N D patients (Table I). For electron microscopic examination, the Correspondence: S. Sasaki, Department of Neurology, Neurological Institute, Tokyo Women's Medical College, Tokyo, 162, Japan. Fax: (81) 3 5269 7324.
lumbar (L1_5) levels o f the spinal cords were fixed in 2% glutaraldehyde with phosphate buffer (pH 7.40). After fixation, the anterior horns at each level were then sectioned transversely, postfixed in 1% osmium tetroxide for several hours, dehydrated, and embedded in epoxy resin. Each block was cut into serial semi-thin sections (about 1 p m thick). These sections were stained with Toluidine blue. Appropriate portions of the sections were cut into ultrathin sections. These were then stained with uranyl acetate and lead citrate. The same procedure was applied to 11 control patients (aged 44-81 years; mean age 59.5 years) who died without having any known neurological disease. Autopsy was performed on all M N D and control patients within 3 h of death. Electron microscopy showed that SI consisted basically of a bundle of compactly packed filaments running parallel to the longitudinal axis without fine granules (Fig. 1). Each filament measured approximately 15-25 nm in diameter and was tubular on transverse sections. A bundle of these grouped tubular filaments was often surrounded by a unit membrane (Fig. 1). SI occasionally appeared to contain electron-dense material (Fig. 2). Sometimes SI showed a combination of fine filaments (neurofilaments) and thicker linear structures (tubules) (Fig. 3). LBHI were composed of randomly arranged filaments associated with fine granules. In addition to SI and LBHI, we often observed in-between or LBHI-like structures (Figs. 4-6). They consisted of dense linear
122 TABLE I NEUROPATHOLOGICAL FINDINGS SI, skein-like inclusion; LBHI, Lewy body-like hyaline inclusion; BB, Bunina body; -, absent; +, mild; ++, moderate; +++, high. Frequency of appearance
Case Age/sex Clinical Degreeof No. course anterior (months) horn cell loss
1 2 3 4 5 6 7 8 9 10 lI
75/M 83/M 49/M 62/M 67/F 67/M 49/M 62/M 63/F 64/M 78/F
6 10 16 22 24 28 29 30 42 65 68
+ + ++ + + ++ + ++ + + ++
SI
LBHI
BB
++ +++ + ++ + +++ + ++ ++ ++
+++ +++ + + + +++ + + +
+ +++ + ++ ++ ++ + ++ +++ ++
structures, frequently c o n t a i n i n g finer filaments (intermediate filaments) (Figs. 4 a n d 5) a n d occasionally elect r o n - d e n s e material; some of them closely resembled BB that consist of electron-dense g r a n u l a r or a m o r p h o u s material frequently with vesicles a n d t u b u l a r structure (Fig. 5), a n d m o r e rarely h a d large electron-dense central cores (Fig. 6). W h e r e the r a n d o m l y - a r r a n g e d thicker filam e n t s were clustered, there were also scattered clumps of electron-dense g r a n u l a r or a m o r p h o u s material closely resembling BB with vesicles, a n d these were sometimes m i n g l e d with the thicker filaments (Fig. 7). SI were freq u e n t l y observed at the periphery o f or inside L B H I , a n d even s u r r o u n d e d some of the L B H I (Fig. 8). T u b u l a r
Fig. 1. A skein-like inclusion consists of bundles of compactly packed filaments running parallel to the longitudinal axis without fine granules. Each filament is approximately 15-25 nm in diameter, x 51,300.
Fig. 2. A skein-like inclusion appears to contain electron-dense material. × 51,300.
profiles were sometimes seen in the L B H I o n cross-sections. SI were m u c h more c o m m o n t h a n L B H I in the anterior horn neurons. The control subjects showed n o b u n d l e s of filaments c o r r e s p o n d i n g to SI in their a n t e r i o r h o r n n e u r o n s . It remains controversial whether SI have any relationship to BB. Recently, M u r a y a m a et al. [11] reported the existence of BB-related structures consisting of b u n d l e s of coated filaments (12 n m in diameter) as possible precursors of BB, a n d these a p p e a r to be identical to the b u n d l e s of t u b u l a r filaments detected in the present study. O n the other h a n d , there are some reports that oppose this conclusion. It has been p o i n t e d out that SI differ from BB b o t h electron microscopically a n d imm u n o c y t o c h e m i c a l l y [9, 1 1, 14]. U n l i k e SI, BB consist of electron-dense material with vesicles a n d t u b u l a r structures, a n d occasionally c o n t a i n cytoplasmic organelles such as n e u r o f i l a m e n t s a n d m i t o c h o n d r i a [12]. I n addition, SI are u b i q u i t i n - p o s i t i v e a n d BB are u b i q u i t i n - n e g -
Fig. 3. A skein-likeinclusion shows a combination of fine filaments and thicker linear structures, x 51,300.
123
Fig. 4. An in-between structure of SI and LBHI which is composed of bundles of thicker filaments containing finer filaments (neurofilaments), x 51,300.
Fig. 5. An in-between structure of SI and LBHI (or LBHI-like structure) which consists of bundles of thicker filaments containing finer filaments (intermediate filaments) and electron-dense material resembling Bunina bodies, x 19,000.
Fig. 7. Electron-dense material closely resembling Bunina bodies with vesicles is scattered among clusters of skein-like inclusions, x 15,200.
ative [3, 14]. In the present study, m a n y SI were indeed f o u n d at separate locations f r o m the BB. However, SI occasionally contained electron-dense material similar to BB, and electron-dense material closely resembling BB were not u n c o m m o n l y mingled with SI. These findings suggest a close relationship between SI and the f o r m a tion o f BB. The relationship between SI and L B H I has also been debated. Some investigators report that the frequency o f SI is not correlated with the presence or absence o f L B H I [9, 11]. However, the present study frequently showed in-between structures and the coexistence o f SI and L B H I . Tubular profiles ( c o m p o n e n t o f SI) were sometimes observed in L B H I , and SI were far m o r e frequently seen c o m p a r e d with L B H I . These findings, taken together with the fact that ubiquitin-positive SI are occasionally connected with ubiquitin-positive L B H I in imm u n o c y t o c h e m i c a l studies [3, 13, 14], seem to indicate a close relationship between these two structures including a possibility that SI m a y be the precursor o f L B H I .
Fig. 6. A LBHI-like structure containing a large central electron-dense core. × 19,000. Fig. 4-6. In-between structures of skein-like inclusions (SI) and Lewy body-like hyaline inclusions (LBHI), or LBHI-like structures.
Fig. 8. Coexistence of LBHI and skein-like inclusions, x 9,500.
124 1 Hirano, A., Kurland, L.T. and Sayre, G.P., Familial amyotrophic lateral sclerosis. A subgroup characterized by posterior and spinocerebellar tract involvement and hyaline inclusions in the anterior horn cells, Arch. Neurol., 16 (1967) 232-243. 2 Hirano, A., Nakano, I., Kurland, L.T., Mulder, D.W., Holley, P.W. and Accomanno, G., Fine structural study of neurofibrillary changes in a family with amyotrophic lateral sclerosis, J. Neuropathol. Exp. Neurol., 43 (1984) 471-480. 3 Kato, T., Katagiri, T., Hirano, A., Kawanami, T. and Sasaki, H., Lewy body-like hyaline inclusions in sporadic motor neuron disease are ubiquitinated, Acta Neuropathol., 77 (1989) 391-396. 4 Leigh, P.N., Anderton, B.H., Dodson, A., Gallo, J.-M., Swash, M. and Power, D.M., Ubiquitin deposits in anterior horn cells in motor neuron disease, Neurosci Lett., 93 (1988) 197-203. 5 Leigh, P.N., Whitwell, H., Garofalo, O., Buller, J., Swash, M., Martin, J.E., Gallo, J.-M., Weller, R.O. and Anderton, B.H., Ubiquitinimmunoreactive intraneuronal inclusions in amyotrophic lateral sclerosis, Brain, 114 (1991) 775-788. 6 Lowe, J., Lennox, G., Jefferson, D., Morrell, K., McQuire, D., Gray, T., Landon, M., Doherty, F.J. and Mayer, R.J., A filamentous inclusion body within anterior horn neurones in motor neurone disease defined by immunocytochemical localization of ubiquitin, Neurosci. Lett., 94 (1988) 203-210. 7 Martin, J.E., Swash, M. and Schwartz, M.S., New insights in motor neuron disease, Neuropathol. Appl. Neurobiol., 16 (1990) 97-110. 8 Matsumoto, S., Hirano, A. and Goto, S., Ubiquitin-immunoreactive filamentous inclusions in anterior horn cells of Guamanian amyotrophic lateral sclerosis, Acta Neuropathol., 80 (1990) 233 238,
9 Mizusawa, H., Nakamura, H., Wakayama, I., Yen, S.-H. and Hirano, A., Skein-like inclusions in the anterior horn cells in motor neuron disease, J. Neurol. Sci., 105 (1991) 14-21. 10 Murayama, S., Ookawa, Y., Mori, H., Nakano, I., Ihara, Y., Kuzuhara, S. and Tomonaga, M., Immunocytochemicaland ultrastuctural study of Lewy body-like hyaline inclusions in familial amyotrophic lateral sclerosis, Acta Neuropathol., 78 (1989) 143152. 11 Murayama, S., Mori, H., Ihara, Y, Bouldin, T.W., Suzuki, K. and Tomonaga, M., Immunocytochemical and ultrastructural studies of lower motor neurons in amyotrophic lateral sclerosis, Ann. Neurol., 27 (1990) 137-148. 12 Sasaki, S., Hirano, A., Donnenfeld, H. and Nakano, I., An electron microscopic study of Bunina bodies, Neurol. Med., (Tokyo) 19 (1983) 317-324. 13 Sasaki, S., Yamane, K., Sakuma, H. and Maruyama, S., Sporadic motor neuron disease with Lewy body-like hyaline inclusions, Acta Neuropathol., 78 (1989) 555-560. 14 Sasaki, S. and Maruyama, S., Immunocytochemical and ultrastrucrural studies of hyaline inclusions in sporadic motor neuron disease, Acta Neuropathol., 82 (1991) 295-301. 15 Schiffer, D., Autilio-Gambetti, L., Chio, A., Gambetti, P., Giordana, M.T., Gullotta, F., Migheli, A. and Vigliani, M.C., Ubiquitin in motor neuron disease: study at the light and electron microscope, J. Neuropathol. Exp. Neurol., 50 (1991) 463-473.
